No abstract

Tissue-wide coordination of polarized cytoskeletal organization and cell behaviour, critical for normal development, is controlled by asymmetric membrane localization of non-canonical Wnt/planar cell polarity (PCP) signalling components. Understanding the dynamic regulation of PCP thus requires visualization of these polarity proteins in vivo. Here we utilize CRISPR/Cas9 genome editing to introduce a fluorescent reporter onto the core PCP component, Vangl2, in zebrafish. Through live imaging of endogenous sfGFP-Vangl2 expression, we report on the authentic regulation of vertebrate PCP during embryogenesis. Furthermore, we couple sfGFP-Vangl2 with conditional zGrad GFP-nanobody degradation methodologies to interrogate tissue-specific functions for PCP. Remarkably, loss of Vangl2 in foxj1a-positive cell lineages causes ependymal cell cilia and Reissner fiber formation defects as well as idiopathic-like scoliosis. Together, our studies provide crucial insights into the establishment and maintenance of vertebrate PCP and create a powerful experimental paradigm for investigating post-embryonic and tissue-specific functions for Vangl2 in development and disease.

Epithelial patterning in the developing eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. mutants have rough eyes and retinal patterning defects that resemble mutants. retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in mutants. However, mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.

Plastids in the viridiplantae sporadically form thin tubules called stromules that increase the interactive surface between the plastid and the surrounding cytoplasm. Several recent publications that report observations of certain proteins localizing to the extensions have then used the observations to suggest stromule-specific functions. The mechanisms by which specific localizations on these transient and sporadically formed extensions might occur remain unclear. Previous studies have yet to address the spatiotemporal relationship between a particular protein localization pattern and its distribution on an extended stromules and/or the plastid body. Here, we have used discrete protein patches found in several transgenic plants as fiducial markers to investigate this relationship. While we consider the inner plastid envelope-membrane localized protein patches of the GLUCOSE 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR1 and the TRIOSE-PHOSPHATE/ PHOSPHATE TRANSLOCATOR 1 as artifacts of fluorescent fusion protein over-expression, stromule formation is not compromised in the respective stable transgenic lines that maintain normal growth and development. Our analysis of chloroplasts in the transgenic lines in the Arabidopsis Columbia background, and in the arc6 mutant, under stromule-inducing conditions shows that the possibility of finding a particular protein-enriched domain on an extended stromule or on a region of the main plastid body is stochastic. Our observations provide insights on the behavior of chloroplasts, the relationship between stromules and the plastid-body and strongly challenge claims of stromule-specific functions based solely upon protein localization to plastid extensions.One sentence summaryObservations of the spatiotemporal relationship between plastid envelope localized fluorescent protein fusions of two sugar-phosphate transporters and stromules suggest a stochastic rather than specific localization pattern that questions the idea of independent functions for stromules.