Browsing by Subject "Planar cell polarity"

Now showing 1 - 3 of 3

Control of intraflagellar transport : studies of the planar cell polarity effector Fuz, the small GTPase Rsg1, and the novel protein TTC29
(2014-05) Brooks, Eric Robert; Wallingford, John B.
Cilia are small microtubule based protrusions found on most cells of the vertebrate body. In humans, defects in the structure or function of cilia results in a large class of developmental and homeostatic diseases known collectively as the ciliopathies. Ciliogenesis is accomplished by the concerted action of a number of molecular pathways including the intraflagellar transport (IFT) system. IFT is a group of ~20 highly conserved proteins that assemble into large macromolecular complexes known as trains. These trains act to carry cargo bi-directionally between the cell body and ciliary tip, via interaction with the microtubule motors kinesin and dynein. IFT train dynamics are required for both cilia structure and function, however the controls on these dynamics are still incompletely understood. Here, I present the first platform for study of IFT dynamics within vertebrate multiciliated cells, an understudied population with critical functions in development and homeostasis. Using this platform, I demonstrate that the planar cell polarity effector protein Fuz is required for IFT dynamics via its control of the cytoplasmic localization of a subset of IFT proteins. Subsequently, I find that a Fuz binding partner, the putative small GTPase Rsg1, is also required for IFT protein localization and dynamics. Additionally, I describe a role for Rsg1 in basal body docking, one of the earliest events of ciliogenesis. Finally, I show that the poorly studied protein TTC29 is required for a specific subset of IFT dynamic behaviors. These data reveal novel regulatory motifs for ciliogenesis and demonstrate, specifically, the complexities of IFT regulation in the cytoplasm and within the cilium itself. Finally, they suggest that multiciliated cells provide a tractable platform for generating robust datasets for the investigation ciliary dynamics. Such studies are critical for informing our understanding of the molecular etiology of human ciliopathic diseases.
Dynamic control of the actin cytoskeleton during convergent extension of the vertebrate embryo
(2022-05) Devitt, Caitlin Collins; Wallingford, John B.; Alvarado, José; Vokes, Steve; Dickinson, Dan; Stachowiak, Jeanne; Ehrlich, Lauren
Tissue morphogenesis requires specific signaling pathways to govern precise behavior of individual cells and the collective tissue. This is exemplified by the requirement of Planar Cell Polarity (PCP) signaling to govern convergent extension (CE) and tissue elongation in model organisms and humans. During this collective cell movement, cells must rapidly rearrange the cytoskeleton, establish anteroposterior polarity, and intercalate between one another. Despite their importance during CE, how PCP proteins function during this dynamic process remains unclear. Importantly, PCP proteins and actin regulators have overlapping functions during this process, and the overarching goal of my thesis was to understand how PCP proteins interact with the cytoskeleton to govern CE. My first project defined the in vivo interactome of Cofilin during CE, identified a role for it’s cofactor Twinfilin in CE, and laid the groundwork for further mechanistic studies of actin turnover during Xenopus convergent extension (Chapter 2). In parallel, I investigated the localization of PCP proteins, found anterior PCP proteins tightly associate with and are required to remodel the actin cytoskeleton into actin rich node-and-cable structures, forming a planar polarized stiffness gradient during cell intercalations (Chapter 3). And through collaboration with the Marcotte lab, we have defined the interactome of 2 polarity proteins, Vangl2 and Dishevelled2, during CE, and identified interactions between PCP proteins and the cytoskeleton, among other things (Chapter 4). Together, by combining tissue-specific proteomics and advanced imaging approaches we have furthered our understanding of the molecular processes governing cellular movements in CE and provided new insights into the basic biology of vertebrate development.
Mechanisms of planar cell polarity patterning by Prickle and Vangl and the control of collective cellular behaviors during tissue morphogenesis
(2017-08) Butler, Mitchell Tyler; Wallingford, John B.; Eberhart, Johann K.; Ehrlich, Lauren I. R.; O'Halloran, Theresa J.; Stachowiak, Jeanne C.
Planar Cell Polarity (PCP) signaling establishes asymmetric molecular patterns that control polarized cellular behaviors within the plane of a tissue or organ. This feature is conserved among metazoans and essential for proper development and tissue homeostasis. Owing to its important role in establishing tissue function, PCP signaling defects have been associated with diverse human pathologies, most notably birth defects. Tissue development is a dynamic process that requires the coordinated, temporal control of polarized cell behaviors, and here, I present the first vertebrate platform for the study of asymmetric PCP tissue patterning dynamics in vivo. Using Xenopus laevis as a model allows for relatively rapid and highly tractable studies of PCP patterning function in developing tissues, affording both subcellular analysis of PCP protein dynamics and multiple readouts for various planar- polarized cellular and tissue behaviors. I identified specific members of core PCP gene families that serve as faithful reporters for molecular planar polarity in the multiciliated epidermis and neural plate epithelia and uncover an essential role for Pk2 in the asymmetric PCP patterning of both of these tissues. I demonstrate that Pk2 function influences the progressive anterior localization of Dvl1 and posterior localization of Vangl1 in the epidermis, which when disrupted, results in severe ciliary orientation defects. Structure-function analysis reveals the conserved domains essential for Pk2 and Vangl1 asymmetry here. After characterizing the dynamic behaviors of apical cell-cell junctions that mediate convergent extension movements in the Xenopus neural plate, I also show that Pk2 and Vangl2 are dynamic and increasingly asymmetrically enriched at shrinking cell-cell junctions during cellular rearrangements. I present evidence that suggests the polarized enrichment of Pk2 promotes the polarized accumulation of the actomyosin contractional machinery that facilitates mediolateral cell intercalations. Lastly, I detail how PCP point mutant alleles identified in human neural tube defect patient studies impact polarized protein localization behavior. Together, these findings establish a robust in vivo platform for the quantitative study of vertebrate PCP signaling and demonstrate the potential of this platform for furthering our understanding of dynamic tissue patterning processes and the molecular etiology of human birth defects.