Devitt CC et al. (2024), PCP and Septins govern the polarized organizati...

XB-ART-60521

Curr Biol 2024 Feb 05;343:615-622.e4. doi: 10.1016/j.cub.2023.12.025.

Show Gene links Show Anatomy links

PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension.

Devitt CC , Weng S , Bejar-Padilla VD , Alvarado J , Wallingford JB .

???displayArticle.abstract???
Convergent extension (CE) requires the coordinated action of the planar cell polarity (PCP) proteins1,2 and the actin cytoskeleton,3,4,5,6 but this relationship remains incompletely understood. For example, PCP signaling orients actomyosin contractions, yet actomyosin is also required for the polarized localization of PCP proteins.7,8 Moreover, the actin-regulating Septins play key roles in actin organization9 and are implicated in PCP and CE in frogs, mice, and fish5,6,10,11,12 but execute only a subset of PCP-dependent cell behaviors. Septin loss recapitulates the severe tissue-level CE defects seen after core PCP disruption yet leaves overt cell polarity intact.5 Together, these results highlight the general fact that cell movement requires coordinated action by distinct but integrated actin populations, such as lamella and lamellipodia in migrating cells13 or medial and junctional actin populations in cells engaged in apical constriction.14,15 In the context of Xenopus mesoderm CE, three such actin populations are important, a superficial meshwork known as the "node-and-cable" system,4,16,17,18 a contractile network at deep cell-cell junctions,6,19 and mediolaterally oriented actin-rich protrusions, which are present both superficially and deeply.4,19,20,21 Here, we exploited the amenability of the uniquely "two-dimensional" node and cable system to probe the relationship between PCP proteins, Septins, and the polarization of this actin network. We find that the PCP proteins Vangl2 and Prickle2 and Septins co-localize at nodes, and that the node and cable system displays a cryptic, PCP- and Septin-dependent anteroposterior (AP) polarity in its organization and dynamics.

???displayArticle.pubmedLink??? 38199065
???displayArticle.pmcLink??? PMC10887425
???displayArticle.link??? Curr Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: prickle2 septin7 vangl2
GO keywords: actomyosin
???displayArticle.antibodies??? Vangl2 Ab7
???displayArticle.morpholinos??? prickle2 MO2 septin7 MO1 vangl2 MO2

???attribute.lit??? ???displayArticles.show???

	Figure 1. PCP protein and Septin localization in actin-rich nodes (A–C) Still images of FP fusions to indicated PCP proteins (green) and Lifeact (magenta) in polarized cells, insets show magnified view of nodes. (D–F) Still images of indicated PCP proteins (green) and Lifeact (magenta) in pre-polarized cells, insets show magnified view of nodes. (G and H) Quantification of the position of FP-labeled PCP foci in polarized and pre-polarized cells (0 indicating anterior, 1 indicating the most posterior limit of the cell). (I–L) Images of indicated Septin subunits (green) and Lifeact (magenta) at nodes; scale bars, 10 μm. (M) Schematic interpretation of data in (A)–(L). (N and O) Vangl2-GFP localization in control and Sept7MO cells. (P) Quantification of AP positioning of Vangl2 puncta. n values and statistics are provided in supplemental information. See also Figures S1–S3.
	Figure 2. Progressive polarization of the actin node and cable system requires PCP and Septins (A–D) Individual cells mosaically labeled with Lifeact-RFP at times indicated at left (maximum intensity projections of cortical actin 10 μm into cell). (A′–D′) Cyan box and inlay highlight individual nodes. Heatmap showing orientation of actin fibers quantified with OrientationJ (legend in A′; cyan, mediolateral; red, anteroposterior). (A′′–D′′) Quantification of actin filament orientation. (E) Quantification of cell length-to-width ratios over time. (F) Upper panel, schematic of AP localization, arrow representing cellular anterior-posterior axis. Lower panel, quantification of Lifeact-RFP labeled nodes over time. (G–K) Representative images of cells with indicated manipulations (maximum intensity projection of cortical actin 10 μm into cell). Inlay shows actin node and cable, where present, indicated by magenta box). (G′ and G′′) Schematics of quantification. (H′–K′) Quantification of actin fiber alignment in cells. (H′′–K′′) Quantification of actin cable positioning. Scale bars, 10 μm. n values and statistics are provided in supplemental information.
	Figure 3. PCP- and Septin-dependent anteroposterior pattern in the node and cable system (A) Representative TIRF image of a control cell, outlined in pink. Boxes highlighting anterior (cyan) and posterior (yellow) regions of the cell. (B and C) Representative anterior and posterior cropped ROIs from (A). (B′–C′) tSOAX segmentation. (B′′–C′′) Representative single actin filament traces over time used for quantification. (D) Quantification of persistence length (Lp) of actin filaments in anterior (A) or posterior (P) cellular regions of control, Sept7MO, and Vangl2MO cells. (E and F) Lifeact-RFP labeling the actin cortex in control and Sept7 MO cells. Boxes indicate regions used for kymograph, below. (E′ and F′) Kymograph of representative actin fiber coalescence over time. (G) Quantification of actin filament coalescence over time, 0 s indicates the full coalescence to a single fiber. Scale bars, 10um. n values and statistics are provided in supplemental information. See also Figure S4.
	Figure 4. Sept7 controls local dynamics of polarized actin flows (A) TIRF image of a single cell labeled with Lifeact-RFP, Pk2-GFP, and actin-647 speckles. (B–D) Zoomed, split channels of the boxed region in (A). Orange and purple boxes indicate regions quantified in (I), below. (E) Heatmap and vector diagram of PIV data for the region shown in (A). Arrows indicate the vector and direction of flow; the legend shows heatmap indicates positive divergence of flow (yellow) or negative divergent (i.e., convergence, blue). (F) Zoomed view of the boxed region of the heatmap in (E). (G) Line plot of Pk2-GFP fluorescence intensities in multiple cells. (H) Line plot showing vector divergence along the same line from which Pk2 intensity was derived, above. Note maximal vector convergence is coincident with the peak of Pk2 intensity (i.e., in nodes). (I) Quantification of actin speckle lifetimes in regions indicated by boxes in (B) and (D), above. n values and statistics are provided in supplemental information.
	Figure S1: Three distinct actin populations contribute to CE in Xenopus gastrula dorsal mesoderm cells. Related to Figure 1. Schematic at left shows a cell in the DMZ with axes labelled, brown and orange structures represent mediolaterally oriented lamellipodia at the superficial and deep level, respectively. Images at right show optical sections through different z-levels of a representative DMZ cell. Actin is labeled by mosaic expression of LifeAct, note anteriorly biased superficial actin cable.
	Figure S2: A-C: Endogenous Vangl2 localizes to ac6n-rich nodes and effect of Vangl2 loss on Sept7. Related to Figure 1. Immunostaining of Vangl2 (green) along with a’) phalloidin (magenta), and DAPI (cyan) showing co-localization of endogenous Vangl2 with ac(n-rich nodes. B. Endogenous Vangl2 in tissue with mosaic morpholino-mediated KD of Vangl2 (outlined in blue), arrowhead pointing to Vangl2 puncta on control side which are absent in the KD condi(on. C. Quantification confirming mosaic knockdown of Vangl2. N=177 control cells from 3 embryos; n= 219 Vangl2 MO cells from 3 embryos, compared by t test. D. Representative image of Sept7-GFP in a control cell; merged view of sept7 (green) and Lifeact (magenta) is shown below. E. Representative image of Sept7-GFP in a Vangl2 KD cell; merged view of sept7 (green) and Lifeact (magenta) is shown below. F. Quantification of Sept7-GFP pixel intensity in control and Vangl2 MO cells. N=102 control cells from 7 embryos; n= 101 Vangl2 MO cells from 6 embryos, compared by t test.
	Figure S3: Septins localize to ac6n-rich nodes. Related to Figure 1. A. Schematic of a septin hetero-octomer. B. Table of combinatorial septin subtypes, bolded subunits are studied in this paper. C. Schematic of representative cell reflecting images in panels D-G. D-G. Representative images of localization of indicated GFP-fused septins; merged view of septins (green) and LifeAct are shown below. c’. Schematic of zoomed view reflecting images in d’-g’. d’-g’. Zoomed view of indicated septins at actin-rich nodes; merged view of septins (green) and Lifeact are shown below. d”-g”. Line intensity plots of Septin GFP fusions and Lifeact intensity across multiple samples. Scale bars=10um. H-J. Representative images of localization of indicated GFP-fused proteins; merged view of the proteins (green) and Lifeact (magenta) are shown below.
	Figure S4: TIRF and SOAX images for Sept7 and Vangl2 MO cells. Related to Figure 3. A-D. Representative anterior and posterior cropped regions of interest from TIRF movies of Septin7MO or Vangl2MO cells as indicated. a’-d’. tSOAX segmentation. a”-d”. Representative single actin filament traces used for quantification.

References [+] :

Butler, Planar cell polarity in development and disease. 2017, Pubmed